Brequinar is a quinoline carboxylic acid described in U.S. Pat. No. 4,680,299, issued Jul. 14, 1987. The term "brequinar" as used herein and in the claims refers to brequinar sodium, which is the compound 2-(2'-fluoro-1,1'-biphenyl-4-yl )-6-fluoro-3-methyl-4-quinoline-carboxylic acid, sodium salt, or other suitable salts or free acidss thereof, and has the formula: ##STR1## Said quinoline carboxylic acids are potent inhibitors of dihydroorotate dehydrogenase (DHO-DH), the fourth enzyme in the de novo pyrimidine biosynthetic pathway. A structure activity relationship for the inhibition of DHO-DH by the quinoline carboxylic acids has been developed, S-F. Chen et al., Biochem. Pharmacol. 40:709-714 (1990). The effect of the DHO-DH inhibition by the quinoline carboxylic acids is to deplete the plasma uridine concentrations in animals and patients, G. J. Peters et al., Cancer Res. 50:4644-4649 (1990). Said quinoline carboxylic acids were shown to be useful as tumor inhibiting agents (U.S. Pat. No. 4,680,299) and also have been shown to be anti-inflammatory agents (U.S. Pat. No. 4,861,783) .
Recently, brequinar was shown to be a potent immunosuppressive agent in vivo to prevent the rejection of transplanted organs (U.S. Pat. No. 4,968,701) with the capability of suppressing 1) mouse heart allograft rejection, M. P. Murphy and R. E. Morris, Med. Sci. Res. 19:835-836 (1991), 2) rat heart, liver, and kidney allograft rejection, D. V. Cramer et al., Transplantation 53:303-308 (1992), and 3) hamster-to-rat-cardiac xenografts, D. V. Cramer et al., Transplantation Proc. 24: 720-721 (1992) . Brequinar is currently undergoing clinical trials for the prevention of graft rejection in transplant patients. Moreover, it has been determined that an analog of brequinar, 2-(1,1'-biphenyl-4-yl)-5-chloro-3-methyl-4quinolinecarboxylic acid, sodium salt, which was also described in U.S. Pat. No. 4,680,299, issued Jul. 14, 1987, is also active as an immunosuppressive agent.
The compound, 2-(2'-fluoro-4'-hydroxy-1,1'-biphenyl-4-yl)-6-fluoro-3-methyl-4-quinoline- carboxylic acid, sodium salt, was the primary metabolite identified from patients treated with brequinar. It was determined that this hydroxyl-metabolite is pharmacologically inactive as an immunosuppressive agent.
While a certain level of brequinar or other active quinoline carboxylic acids must be maintained in the bloodstream to prevent rejection of transplanted organs, distribution and metabolism of these compounds can vary between individuals as well as between measurements within a single individual during the course of therapy. Accordingly, it is important to monitor the level of quinoline carboxylic acids in biological fluids in a rapid and convenient manner to maintain and adjust dosage for proper pharmacodynamics in transplant patients. Measuring brequinar and other active quinoline carboxylic acids in clinical samples from patients is an important tool to enable one to assure maximum efficacy of the drug. Such measurement in samples could potentially be complicated by the presence of inactive metabolites of quinoline carboxylic acids therein, such as the inactive hydroxyl-metabolite mentioned above. Therefore, it is also desirable to be able to measure brequinar or other quinoline carboxylic acids independently from any inactive metabolites thereof. This invention is directed to antibodies and other reagents, immunoassays and diagnostic kits useful for the measurement of quinoline carboxylic acids present in biological fluids from patients treated with said quinoline carboxylic acids.
Immunoassays are often used for therapeutic drug monitoring because of their rapidity, convenience and sensitivity. In particular, the immunosuppressant, cyclosporine A, is widely monitored using a variety of immunoassays, U. Kunzendorf et al., Klin Wochenschr 67:438-441 (1989) and B. Tjandra-Maga et al., J.Clin Chem Clin Biochem 28:53-57 (1990). EPO Application No. 473,961 (Morrison, et al.) describes immunoassay reagents and a method for determining cyclosporine in test samples. Presently, brequinar is measured by high performance liquid chromatography, which is very costly and time-consuming. Nevertheless, there remains the need for specific high affinity reagents and immunoassays to quickly and acurately measure quinoline carboxylic acid levels, including brequinar, in patients. The present invention is directed to addressing these, as well as other, important needs.